About 3% of the whole genome, the basis for protein-coding genes, are targeted by WES, while exome and targeted sequencing offer a balance between cost and benefit, compared with whole genome sequencing (WGS) [21]. However, the molecular etiology is still limited to confirm the genetic heterogeneity in the disease of OSL. Whole exome sequencing implicates PTCH1 and COL17A1 genes in OPLL of the cervical spine in Chinese patients [15]. A whole-exome sequencing approach defines the molecular signatures of anterior cruciate ligament ruptures and Achilles tendinopathy [22]. In the present study, we selected 5 patients with typical radiological findings of OSL and 5 control patients to allow description of genetic variants related with OSL through WES approach.
We detected 8 common SNP variants in all 5 subjects of OSL. These variants were generated in the genes of GRHL2, CUL3, WHAMM, IL17RD, POM121L12, SLC26A8 and PTPN23. Furthermore, among that, two novel variants were identified in WHAMM, that were c.A2207C and c.C2208G, while their corresponding protein changes were p.H736P and p.H736Q. These recognized genes may participate in pathologic progression of OSL. WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is one member of Wiskott-Aldrich syndrome protein (WASP) family, which functions for the ubiquitously expressed Arp2/3 complex as nucleation-promoting factors [23]. WHAMM links actin assembly to autophagy through the Arp2/3 complex [24]. WHAMM promotes actin polymerization to initiate autolysosome tubulation on autolysosomes [25]. An actin WHAMM interaction links autophagy and SETD2 [26]. Rab1 recruits WHAMM but limits actin nucleation during membrane remodeling [27]. Autophagy is involved in the osteogenic differentiation of ligament fibroblasts and promotes the development of OPLL [28]. Therefore, it is possible that novel variants in WHAMM gene can regulate the action of autophagy to affect osteogenic differentiation in the osteogenic process. Our data, together with these previous studies, imply that WHAMM may be one of important genes attributed to underlying genetic factors in the disease of OSL, whereas more studies need to be further investigated.
In addition, we found 4 common SNP variants were presented in 4 subjects of OSL among 56 common SNP variants when added additional screenings with deleterious Polyphen2_HDIV_score, Polyphen2_HVAR_score and SIFT. The genes are KRT84, KIF1B, NRAP and CETN. The novel variant in the gene of KIF1B was identified as risk factors for disease by the five prediction tools. KIF1B, the kinesin motor protein, has previously been involved in the axonal transport of synaptic vesicles and mitochondria and has been related with susceptibility to multiple sclerosis (MS) [29]. Elevated levels of KIF2A and KIF1B proteins in mature Dendritic cells (mDCs) inhibit lamins degradation, likely by hampering autophagosome-lysosome fusion [30]. IGF1R, the receptor tyrosine kinase, is a new direct binding partner of KIF1Bβ, and its binding and transport is impaired specifically by the Y1087C mutation in KIF1Bβ [31]. IGF1R signaling plays essential roles in self-renewal, pluripotency (or multipotency), and therapeutic efficacy of multiple stem cells [32].MiR-376c-3p reduces IGF1R/Akt signaling in bone marrow-derived stromal cells (BMSC) and is one mechanism by which osteogenesis may be inhibited [33]. Let-7c targets IGF-1R to inhibit the osteo/odontogenic differentiation of dental pulp-derived mesenchymal stem cells (DPMSCs) treated with IGF-1 [34]. Mesenchymal stem cells (MSCs) in human spinal ligaments localize to collagenous matrix and perivascular area, which have a high propensity toward osteogenesis [35]. Based on the previous findings, we hypothesize that novel variants in KIF1B gene may alter osteogenesis progression of mesenchymal stem cells (MSCs) through IGF1R signaling in spinal ligaments. These observations suggest that the novel variants in the gene of KIF1B may contribute to the pathophysiology of OSL.
Besides, we found 7 common SNP variants were presented in 3 subjects of OSL among 207 common SNP variants when added additional screenings with deleterious Polyphen2_HDIV_score, Polyphen2_HVAR_score and SIFT. The genes are CCT3, ANLN, ESRRB, SRBD1, ODF3L1, BRAT1 and RBP3. The novel variants in the genes of ESRRB and RBP3 were assessed as risk factors for disease in all five prediction tools. During the reprogramming of epiblast stem cells, Esrrb (estrogen-related receptor β), the orphan nuclear receptor, plays a pioneering role in recruiting the core pluripotency factors Sox2, Oct4 and Nanog to inactive enhancers in closed chromatin [36]. Ncoa3 sustains embryonic stem cell self-renewal and reprogramming through functioning as an essential Esrrb coactivator [37]. Esrrb mediates reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells by conjunction with Oct4 and Sox2 [38]. Mutations of ESRRB encoding Esrrb causes autosomal-recessive nonsyndromic hearing impairment DFNB35 [39]. Mesenchymal stem cells (MSCs) in OPLL have a high propensity toward osteogenesis [40]. Osteogenic differentiation is suppressed by a histamine-2-receptor antagonist in MSCs from OPLL patients [41]. MiR-615-3p suppresses osteogenic regulators GDF5 and FOXO1 to inhibit osteogenic differentiation of human lumbar ligamentum flavum cells [42]. Therefore, we speculate that whether ESRRB participates in OSL through regulates the osteogenic differentiation in MSCs, which needs to be validated in the future study. To our knowledge, there are limited studies on the RBP3 gene. These results suggested that protein change induced by a single-nucleotide substitution in the gene of ESRRB could be one potential mechanism affecting OSL in patients.
Our data exhibited that several common SNP variants were only presented in 3 or 4 subjects among the 5 OSL patients. Although ascertaining the exact causes for the discrepancies among these patients is hard, potential contributors to the inconsistent consequences may include study design, sample size, genetic heterogeneity between populations, levels of gene-environment interactions and environmental factors such as mechanical stress [13]. Inclusion standards with more serious phenotypes can promote the genomic research of disease-susceptibility genes through organizing genetically more homogeneous individuals [43].